Recently, biomolecular arrays have been successfully created. For example, Fodor, et al., "Light-directed, Spatially Addressable Parallel Chemical Synthesis," Science, Vol. 251,767-773 (1991) disclose high density arrays formed by light-directed synthesis. The array was used for antibody recognition. Biomolecular arrays are also described by E. Southern (PCT Publication WO 89/10977) for analyzing polynucleotide sequences. Such biomolecular arrays lend themselves to a large number of applications, from DNA and protein sequencing to DNA fingerprinting and disease diagnosis.
Large scale polymer arrays may be needed for specific applications. In the analysis of large polymeric molecules such as the sequencing of DNA and protein identification, because of the large number of possible combinations, the ability to "read" (i.e., detect differences in) the polymer array is important for such analyses and sequencing to be practicable. For example, in DNA sequencing, one may wish to scan an array of all possible 65,000 8-mers (a 8-mer is a single strand polynucleotide having 8 nucleic bases). Each pixel corresponds to a single 8-mer and practical limits on the amount of optical power required to read this pixel are between 1 mW and 10 mW.
One approach for synthesizing a polymer array on an optical substrate is described by Fodor et at. (1991) supra; PCT publications WO 91/07087, WO 92/10587, and WO 92/10588; and U.S. Pat. No. 5,143,854. Because the apparatus and method of synthesizing a polymer array can be applied in the present invention, these disclosures are incorporated by reference herein. In this approach, an array of different receptors is synthesized onto a substrate using photolithographic techniques. Ligands are washed over the array. Either the ligand is fluorescently labeled or an additional fluorescently labeled receptor is also washed over the array. The result is that fluorophores are immobilized on those pixels where binding has occurred between the ligand and the receptor(s). The array is illuminated with radiation that excites the fluorophores. The pattern of bright and dark pixels is recorded. Information about the ligand is obtained by comparing this bright-dark pattern with known patterns of surface bound receptors. The aforementioned references describe a method for reading the array for the presence of fluorophores. For example, PCT publication WO 92/10587 discloses optically scanning an array by directing excitation with light through a microscope objective and collecting fluorescence through the same objective. All the embodiments described in the PCT Publication WO 92/10587 refer to similar direct illumination of the array. Similarly, PCT Publication WO 92/10092 discloses directly illuminating the array surface in all its embodiments.
While direct illumination of the array is simple, there are some significant disadvantages. For example, excitation radiation reflected from the array surface can enter the fluorescence-collection optics. This reflected radiation can be much brighter than the generated fluorescence. Direct illumination almost always results in excitation and scattering from a large number of molecules other than the fluorophores, additionally creating a possibly larger background signal. This is particularly problematic if the illuminating light passes through a solution in contact with the array. While there may be techniques to reduce these effects (such as temporal, spectral or spatial filtering of the light), their utility is often limited by trade-offs that occur between signal-to-noise and collection time.
Optical-frequency evanescent probing has been investigated for chemical assay. Evanescent surface probing techniques using fluorescence include total internal reflection on prisms (M. L. Kronick and W. A. Little, "A new immunoassay based on fluorescence excitation by internal reflection spectroscopy," J. Immunological Meth., Vol. 8, 235, 1975), waveguides (A. N. Sloper, J. K. Deacon, and M. T. Flanagan, "A planar indium phosphate monomode waveguide evanescent field immunosensor," Sensors and Actuators, Vol. B1, 589, 1990), and optical fibers (R. P. H. Kooyman, H. E. be Bruijn, and J. Greve, "A fiber-optic fluorescence immunosensor," Proc. Soc. Photo.-Opt. Instrum. Eng., Vol. 798, 290, 1987); and surface plasmon resonance (J. P. Seher, "Method and apparatus for detecting the presence and/or concentration of biomolecules, "European Patent No. 0 517 930 A1, 1992).
In such evanescent probing techniques, high contrast is achieved against reflected and scattered excitation radiation because the excitation energy does not travel through space but is trapped in a very thin region above the surface. Additionally, the 1/e depth (i.e., the depth for the attenuation of light to 1/e of the original value) of this region may be controlled (P. Lorrain and D. Corson, Electromagnetic Fields and Waves, W. H. Freeman, San Francisco, 1970, pp. 520-525).
Evanescent excitation is very different from direct excitation or direct illumination as described in PCT publications WO 92/10587 and WO 92/10092. Gratings are often used to convert direct illumination into evanescent excitation (D. S. Goldman, P. L. White, and N. C. Anheier, "Miniaturized spectrometer employing planar waveguides and grating couplers for chemical analysis, "Appl. Opt., Vol. 29, 4583-4589, 1990). It is well known in the art that gratings may also be generated by acoustic and optical waves and have been used to create evanescent excitation (X. Sun, S. Shiokawa, and Y. Matsui, "Interactions of surface plasmons with surface acoustic waves and the study of the properties of Ag films," J. Appl. Phys., Vol. 69, 362, 1991). However, the prior art methods as illustrated in the above publications for scanning arrays typically employ argon ion lasers, which tend to be expensive and large.
To effectively illuminate a large array requires a large amount of energy. For example, illuminating simultaneously all the pixels in the aforementioned array of 65,000 8-mers requires between 65 W and 650 W. Because of the inefficiency in light generation, sources that generate this amount of optical power will consume much more electrical power than these numbers indicate, making cooling necessary. What is needed is an apparatus and a technique that can be used for detecting a polymeric target substance on a polymer array with high contrast against background noise using compact, low power hardware.